Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements

Abstract

The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis CAUTI incidence and severity.

Pathogenesis of Proteus mirabilis Infection

“…The original P. mirabilis sequenced genome, HI4320, included a 36 kb plasmid that was designated pHI4320 (112), and a signature-tagged mutagenesis study as well as a transposon insertion-site sequencing study indicated that mutations in pHI4320 led to decreased fitness in mice (108, 129). However, several of the targeted genes were involved in plasmid stability, and pHI4320 does not encode any obvious virulence genes (112).…”

“…Several variations of the method were developed concurrently, including insertion sequencing (INSeq) (181), transposon-directed insertion site sequencing (TraDIS) (182), transposon insertion-site sequencing (Tn-Seq) (183, 184), and high-throughput insertion tracking by deep sequencing (HITS) (185). As Tn-Seq has been applied to the study of P. mirabilis pathogenicity (129), the specifics of this method will be explained in further detail.…”

“…This approach was successfully used to generate the first genome-saturating library of transposon insertion mutants in P. mirabilis HI4320, and allowed for identification of 436 genes (12% of the genome) estimated to be essential for growth in Luria broth (129). As would be expected, the essential genes largely pertained to cell cycle control and division, cell wall biogenesis, replication, and ribosomal proteins, and 64% had been identified as essential genes in other bacterial species.…”

“…Importantly, this approach was also applied during polymicrobial infection with P. stuartii , in parallel with the above study, to determine how polymicrobial infection influences P. mirabilis fitness requirements (129) (Fig. 15).…”

“…Interestingly, an additional 1,353 candidate fitness factors were identified that appear to specifically contribute to P. mirabilis fitness during polymicrobial infection, including defense mechanisms (such as type III secretion and type VI secretion), signal transduction pathways, at least 10 distinct fimbrial types, iron uptake systems, respiratory nitrate reductase, the oxidative pentose phosphate pathway and the Entner-Doudoroff pathway, arginine import and biosynthesis, and branched chain amino acid (BCAA) biosynthesis. It was further determined that the requirement for BCAA biosynthesis during coinfection was due to high-affinity import of leucine by P. stuartii , indicating the utility of this technique for dissecting polymicrobial interactions during infection (129). …”